The initial biochemical step in the mechanism of action of Epidermal Growth Factor (EGF) and polypeptide hormones in general, is reasoned to be its binding to specific receptors on the cell surface. Our main objective, therefore, will be to investigate EGF-receptor interactions in defined experimental systems. This will require the use of cell free systems in which EGF forms a hormone receptor complex which initiates a defined biochemical reaction so that binding can be coupled to biologic responses. We have chosen as our model the human epidermoid carcinoma cell line, A-431 because it has an extraordinarily high concentration of specific membrane receptors for EGF (2-3 million/cell). We have found that binding of EGF to these membranes in vitro produced a marked immediate stimulation of the phosphorylation of both endogenous and exogenous protein substrates in the presence of (Y-32P) ATP due to a cyclic nucleotide independent, tyrosine residue specific, protein kinase. In A-431 cell membranes two forms of EGF receptor-kinase have been observed (170K, 150K daltons). EGF stimulated kinase co-purifies with EGF receptor as the 150K from isolated membranes but as the 170K from vesicles. An endogenous calcium activated protease generates the 150K from the 170K. Prolonged receptor occupancy and internalization appear to be required for the mitogenic effects of EGF. It is likely that receptor processing and its subsequent proteolysis generate several forms of the EGF receptor-kinase which have altered kinase activity, substrate specificity and linkage to more than one regulatory pathway affecting cellular functions. Therefore, we propose to study: 1) role of specific membrane and cytoplasmic components in initial events occuring after EGF binds to cells or cellular subfractions; 2) role of autophosphorylation in regulating EGF receptor-kinase; 3) structure of EGF receptor-kinase using labeling, selective digestion, peptide mapping and sequence analysis methods; 4) by biochemical and immunological techniques the relationships between normal and abnormal differentiation and EGF receptor-kinase.